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Evaluation of natural diatomaceous earth deposits from South
Eastern Europe for stored-grain protection:
The effect of particle size
Bill J. Vayias1,*, Christos G. Athanassiou1, Zlatko Korunic2 and Vlatka Rozman3
1Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, 75
Iera Odos str., 11855, Athens, Greece
2 Diatom Research and Consulting Inc., 14 Greenwich Dr., Guelph, ON, N1H 8B8, Canada
3Department for Plant Protection, University of Josip Juraj Strossmayer in Osijek, 3 Trg Sv.
Trojstva, 31100 Osijek, Croatia
*Author for correspondence: [email protected]
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Abstract
BACKGROUND: The use of Diatomaceous Earths (DEs) provides a promising alternative to
the use of contact insecticides in stored product IPM. Geographical origin and the physical
properties of a given DE may affect its insecticidal activity. In our study, DE samples were
collected from different locations of south-eastern Europe and their efficacy was evaluated in
the laboratory against Cryptolestes ferrugineus (Stephens) (Coleoptera: Cucujiidae),
Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and Rhyzopertha dominica (F.)
(Coleoptera: Bostrychidae). In addition, three fractions comprising particles of different size
were obtained from each DE sample and assessed with regards their effectiveness against the
above stored-product insect pests.
RESULTS: DE from the Greek region of Elassona was the most effective against C.
ferrugineus and S. oryzae, whereas the DE Kolubara 518, mined in the Serbian region of
Kolubara, was the most effective against R. dominica. Smaller particles were more effective
than the larger particles against the three tested species, although significant differences in the
efficacy of the fractions containing particles of 0-150 microns and particles with sizes <45
microns were not always recorded.
CONCLUSIONS: Deposits from south-eastern Europe appeared to be very effective against
the tested species and, therefore, this region should be further evaluated as a source of
development of commercial products. Moreover, particle size is a physical property that
should always be taken into account during the DE manufacturing process as it can strongly
influence the insecticidal action of a given product.
Key words: diatomaceous earth, particle size, effectiveness, insects, grain protectants
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1. Introduction
Over recent years, there has been an increased interest for the use of diatomaceous
earths (DEs) for the protection of stored-grain against arthropod pests.1 Several DE
formulations are now commercially available in many parts of the word, and are used with
success in stored-products protection.2 DEs are composed of the fossils of phytoplankton
(diatoms) that exists in aquatic ecosystems1. Due to the natural origin and the safety of DEs1
the detection of new, natural deposits, has received significant attention.
DE efficacy against insects is affected by biotic and abiotic factors such as
temperature, relative humidity, commodity type, and insect species and therefore, these
factors need to be taken into account when evaluating DE as a grain protectant. 2-7 Although
the main drawback of the use of DEs as grain protectants is that they reduce bulk density (test
weight) of the grain, it may be possible to mitigate the negative impact on grain properties as
long as the most efficacious DEs are selected and applied when factors that enhance DE
efficacy are prevailing in the storage environment. 1, 2, 8-10
The physical and chemical properties of DE such as the percentage of amorphous
silicon dioxide, pH value, active surface, sorption capacity, particle size distribution,
adherence DE particles to kernel are the critical factors affecting their insecticidal action.1
Although, the geographical origin has a great effect on physical and chemical properties of
DEs11, mapping the DE sources according to our knowledge can not be used to predict the
insecticidal activity of DE against insects. DEs collected from the same locality but taken
from different layers are often different in their insecticidal activity since DE itself is
heterogeneous rock, which is consisted of different diatom species that grew under different
conditions during millions of year1.
In a previous report, Korunic,11 conducted experiments with several DEs from
different countries (USA, Mexico, Australia, Japan, China, Canada, former Yugoslavia), and
noted significant variations among the tested DEs with respect to their insecticidal activity. In
this work, the author noted that one of the best DEs was derived from the Former Yugoslav
Republic of Macedonia. Until present this is the only published paper concerning the
insecticidal activity of DE from South Eastern Europe. Hence, it was indicated that this area is
likely to contain other, equally or more effective, natural DE deposits that should be further
evaluated. The author also suggested that the size of DE particles may be playing important
role in the insecticidal value of a given DE and this finding was the agreement with previous
results of Chiu12 and McLaughlin.13
The objective of the present study was to determine the effectiveness of DEs obtained
from multiple locations in South Eastern Europe as stored-grain protectants, against three
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major stored-product beetle species, the rusty grain beetle, Cryptolestes ferrugineus
(Stephens) (Coleoptera: Cucujiidae), the rice weevil Sitophilus oryzae (L.) (Coleoptera:
Curculionidae), and the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera:
Bostrychidae). In this work, the effect of particle size on the insecticidal efficacy of the tested
DEs was also examined.
2. Materials and Methods
2.1 DE deposits used
A total of 14 DEs was examined, obtained from several locations in Greece, Serbia and
Slovenia, during autumn 2007 (Table 1). For comparative purposes, Silicosec® (Biofa GmbH,
Munchigen, Germany), which is a commercially available DE formulation, was used in the
experiments. Silicosec® is a DE of fresh water origin and contains 91.2% SiO2.
2.2 Preparation of the samples
All DE samples were dried to about 6% moisture content, at 40 oC for 24h 11 and sieved
(dry sieving) with U.S.A. standard testing laboratory sieves with meshes of 150 and 45
microns. The experiments were conducted with a base DE with particles sized between 0-150
microns and 2 fractions; 45 to 150 microns and <45 microns fraction. To obtain the base DE,
each DE sample was sieved using a sieve with openings of 150 microns. The particles with
diameters above 150 microns were not used in bioassays because this fraction usually
contains sands, rocks and only a few very large diatoms. The base DE was again sieved using
a sieve with openings of 45 microns to prepare the additional two fractions of 45-150 microns
and <45 microns. The weight of each fraction was measured and its proportion to the base DE
sample was assessed (Table 1).
2.3 Insect species and Commodity tested
Seven to 21d old mixed-sex adults of C. ferrugineus, S. oryzae, and R. dominica were
used in the bioassays. All three species were laboratory cultured at 27 ± 1 oC and 70 ± 5% r.h.
and held in continuous darkness. Cryptolestes ferrugineus was reared in wheat flour while S.
oryzae and R. dominica were reared on whole wheat. The tested commodity was untreated
hard wheat (var. Mexa) with very little dockage (<0.8%). Prior to the start of the experiments,
the moisture content of the grain, as determined by a Dickey – John moisture meter (Dickey-
John Multigrain CAC II, Dickey-John Co, Lawrence, KS, USA), was approx. 12.2 ± 0.3 %.
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2.4 Bioassays
The various fractions of DE were added to the grain held in jars (15 cm in diameter and
35 cm in height) containing 300 grams of wheat at a rate of 600 ppm. The jars were tightly
sealed with lids and thoroughly shaken by hand for one minute, to achieve the equal
distribution of the DE in the grain mass. Subsequently, the grain from the treatment jars was
divided into a further three glass vials (3 sub-replicates) each containing 100 g of treated
wheat. The dimension of the glass vials was 7.5 cm in diameter and 12.5 cm in height. In
addition, three glass vials containing 100g of untreated grain served as controls. Vials were
subsequently infested with 50 adults of either S. oryzae, R. dominica or C. ferrugineus.
Adults were sieved out the treated grain after 24h and 6 days of exposure and dead individuals
were counted at each exposure interval. Bioassays were carried out at 30 oC and 70 % r.h. The
whole procedure was repeated three times and r.h was maintained using saturated salt
solutions throughout the experimental period as recommended by Greenspan.14
2.5 Data analysis
Although control mortality did not exceed 4% in any experiment, insect mortality was
corrected using Abbott’s15 formula. For each insect species, data were initially analysed
according to the GLM procedure of SAS14 with corrected mortality as the response variable
and exposure interval, tested DE and particle size as the main effects. Then, for a specific
exposure x insect species combination, data were subjected to one-way analysis of variance
(ANOVA) according to the GLM Procedure of SAS.16 Means were separated using the Tukey
- Kramer (HSD) test at P= 0.05.17 To evaluate if differences in mortality caused by the
complete DE fraction (0-150 microns) were correlated with the proportion of complete
fraction made up of particles <45 microns, the Correlation Procedure of SAS16 was used. The
above procedure was used for each species x exposure combination (P<0.05; Ho: ρ= 0).
3. Results
3.1 General results
All main effects and associated interactions for mortality levels of C. ferrugineus were
significant (DE: F13,755= 263.4, P<0.001; particle size: F2,755= 311.7, P<0.001; exposure
interval: F1,755= 5387.2, P<0.001; DE x particle size: F26,755= 11.8, P<0.001; DE x exposure
interval: F13,755= 78.3, P<0.001; particle size x exposure interval: F2,755= 46.3, P<0.001) at
P<0.05 level. This was also the case with mortality levels of S. oryzae (DE: F13,755= 732.1,
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P<0.001; particle size: F2,755= 330.2, P<0.001; exposure interval: F1,755= 27841.3, P<0.001; DE
x particle size: F26,755= 5.3, P<0.001; DE x exposure interval: F13,755= 519.6, P<0.001; particle
size x exposure interval: F2,755= 200.9, P<0.001) as well as with mortality levels of R.
dominica (DE: F13,755= 921.6, P<0.001; particle size: F2,755= 276.1, P<0.001; exposure interval:
F1,755= 3098.1, P<0.001; DE x particle size: F26,755= 16.4, P<0.001; DE x exposure interval:
F13,755= 484.3, P<0.001; particle size x exposure interval: F2,755= 186.7, P<0.001) at the same P
level.
3.2 C. ferrugineus
In the case of C. ferrugineus, efficacy of all 3 fractions of Silicosec was extremely high
(>99%) even after the shortest exposure period (24h). Over this exposure interval and with the
exceptions of (a) the small particles of Elassona 1 and Kriti and (b) the particles sized 0-150
microns of Elassona 1, which produced very high mortality (>90%) against this species,
Silicosec was significantly the most effective among the tested DEs (Table 2). Six days post
treatment, with the exception of Begora, Kolubara 516, Serbia lower and Vranje, which were
significantly less effective than Silicosec, all the remaining DEs were equally effective to
Silicosec (Table 2). Over the same exposure period and in more than 60% of the tested DEs,
the smallest particles were significantly more effective compared to the largest particles of the
same DE, against adults of this species (Table 2).
For both exposure periods a significant positive relation between the DE efficacy against
rusty grain beetle and the proportion of small particles (<45 microns) of the base DE was
observed (r = 0.4514, and 0.3427 for 24h, and 6d respectively; P < 0.0001; n = 122; Fig.1)
although significant differences between the fractions <45 microns and 0-150 microns were
not always recorded (e.g Serbia lower and Vranje) (Table 2)
3.3 S. oryzae
Twenty four hours post treatment of wheat with the tested DEs, only Silicosec gave
significant mortality, which ranged between 21.8% and 22.4%, against rice weevils. Over the
same exposure interval, survival of this species on wheat treated with the remainder DEs was
generally high (>92%). The fraction of Silicosec with the largest particles was the least
effective whereas the fraction with the smallest particles (<45 microns) was the most
effective. However, significant differences in weevil mortality were not noted between the
fractions <45 microns and 0-150 microns (Table 3). Six days post treatment of wheat with the
DEs, effectiveness of DE fractions with small particles was significantly higher than that of
fractions with the largest particles except for the fractions of Silicosec, which were 100%
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efficacious against S. oryzae (Table 3). At the same exposure interval, the lowest mortality
levels were noted in wheat treated with Begora, where less than 20% of weevils were killed,
even with the fraction containing the smaller particles. Of the fractions containing the large
particles (45-150 microns) the most effective belonged to Kolubara 518, where rice weevil
mortality reached 89.6%. By contrast, the most effective of the non-commercial DEs with
particles <45 microns was Kolubara middle (98.2%) followed by Kolubara 518 (97.6%) and
as a result, significant differences between those DEs and Silicosec were not recorded (Table
3). This was the only case that significant differences in efficacy of non-commercial DEs and
Silicosec were not recorded against S. oryzae, since the remaining DEs were consistently less
effective than Silicosec against this species.
For the rice weevil, DE efficacy and the content of each DE in particles less than 45
microns were positively correlated, for both exposure intervals (r = 0.5715, and 0.4080 for
24h and 6d respectively; P < 0.0001; n = 122; Fig.1).
3.4 R. dominica
After 24h of exposure, the efficacy of all fractions of the non commercial DEs was very
low (<2.5%) and only the fractions of Silicosec gave mortality, which was also low and did
not exceed 19% (Table 4). Five days later, significantly more beetles were dead on wheat that
was previously treated with the small-particles of a specific non commercial DE compared to
large particles of the same DE (Table 4). Over the same exposure period, mortality of the
lesser grain borer reached 63.8% on wheat treated with the smaller particles of Elassona 1,
whereas it did not exceed 11% on wheat treated with the largest particles of Begora, Elassona
2, Kolubara 516, Kolubara 517, Kriti, Serbia lower or Vranje (Table 4). All tested non
commercial DEs were significantly less effective than Silicosec against adults of R. dominica,
and this was noted for both exposure periods that were tested here.
DE efficacy against lesser grain borer and the proportion of each DE in particles less
than 45 microns were positively related. Moreover, this fact was noted for both exposure
intervals that were tested (r = 0.5126 and 0.5863 for 24h and 6d respectively; P < 0.0001; n =
122; Fig.1).
4. Discussion
Korunic11, when assessing the impact of several DE characteristics on the insecticidal
value of DEs, stated that DE particles between 1 and 30 microns could be positively related
with high DE efficacy. Results indicated that although particle size distribution in a certain
range (median particle size from about two to 30 microns) was cited in references as a very
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important DE property affecting insecticidal activity11-13, this property alone could not be used
to predict the insecticidal value of different DEs. According to the author's11 findings, some
DE formulations with smaller particles (diameter in microns), gave significantly higher
efficacy against the same species of insect than larger particles (e.g.,“Celite 209") but in the
case of a DE originating from the Former Yugoslav Republic of Macedonia, different
fractions (four fractions and the “normal” formulation) with particles between 0 -192 microns
gave similar insecticidal efficacy. In the present study, although significant differences
between the efficacy of the tested DEs and their fractions with the smallest particles (<45
microns) were not always recorded, our results indicate that particle size is a DE property that
can affect their insecticidal action. In light of our findings, DE particles of <45 microns had
stronger insecticidal properties in comparison with larger particles. This was also supported
by the fact that a significantly positive relationship between the efficacy of the tested DEs and
their contents of small-particles was always recorded in our experiments. DEs with smaller
particles have greater surface to volume ratio in comparison with DEs containing larger
particles. Hence, the higher the content in small particles in a given DE quantity, the larger the
surface it covers and the greater the toxicity it produces since the contact area between
insect’s body and the particles increases.2, 18 Unfortunately, the most active DE formulations
against stored grain insects, such as enhanced Protect-It™, Dryacide®, Insecto®, Celite 209,
Dicalite, and DiaFil, also had the greatest effect on the reduction of bulk density.19,20
Based on our findings, Silicosec, which had the greatest content (90%) of small
particles, gave the highest mortality ratio in all of the cases that were tested here.
Nevertheless, as we mentioned already 11, particle size alone cannot be used to predict the
insecticidal value of a certain DE and our results support this statement. According to our
findings, Kolubara 518 and Serbia-upper had the greatest content (75% and 70% respectively)
of small particles of the non commercial DEs that were tested here. However, these DEs were
not always the most effective against the tested species. For instance, in the case of R.
dominica after 6d exposure, the Elassona 1 (50% of particles sized less than 45 microns), was
significantly more effective than Serbia-upper. Similar results were also obtained in the case
of S. oryzae after a 3d exposure period. In the latter case, DE from Slovenia was much more
effective than Serbia-upper. It is noteworthy that DE from Slovenia had the smallest content
in particles <45 microns (30%) of the DEs that were tested here. These contradictory results
obtained from our experiments can be attributed to the fact that besides particle size, there are
also other DE properties that could affect their insecticidal value such as active surface and oil
adsorption, the diameter of inner pores of particles, moisture content of dust, SiO2 content,
and tapped density. 1,11,18,21-24
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DE is prepared for commercial use by quarrying, drying and milling. Practically, the
only change to DE during this process is the reduction in the moisture content and mean
aggregate particle size. The result of this process is a fine, talc-like dust, with the mean
particle size distribution from 0 to more than 100 microns with the majority from 10 to 50
microns.1 A similar procedure was also followed in our experiments with the aim to obtain the
samples of the non commercial DEs and the different fractions from each sample. However,
DE performance was not equal among the DEs, showing that DE origin, even in the case of
adjacent geographical locations, is also a very important factor that affects DE efficacy. In
light of our findings, Elassona 1 and Kolubara 518 originating from Greece and Serbia,
respectively, seemed to be very promising DEs that can be further assessed as potential DE
formulations. These DEs also had very effective large particles (45-150 microns) which were
in some cases more effective than the smallest particles of the other DEs. Hence, it is possible
to use even the larger particles (45-150 microns) of these DEs, if the process of the production
of particles <45 microns is proves expensive. However, large particles (>150 microns) that
usually contain rocks, sand and very large diatoms should be removed during the formulation
process as they can reduce DE performance. 1,11
To obtain a satisfactory level of efficacy, the current DE formulations should be
applied at doses between 400 and 1000 ppm.5 Nevertheless, at this rate DE application may
reduce grain bulk density (volume/weight ratio) and the commercial value accordingly.
Therefore, the looking for DEs which are effective at low doses is of great importance. Also,
DEs perform better with increasing temperatures, so their combined use with high
temperatures have been shown to be very promising for the control of stored product pests. 2-7
In our experiments, we evaluated all DEs at a moderate dose (600 ppm) and at 30 oC. Our
results were promising for C. ferrugineus and S. oryzae (only after 6d exposure) but not for R.
dominica. Hence, to control this species with the tested non commercial DEs, doses higher
than 600 ppm or temperatures higher than 30 oC are required.
Several DEs, mined out from natural deposits in areas rich to silacaceous rocks, have
been demonstrated to be very effective against stored grain pests and are now commercially
available.2 Although the area of Former Yugoslavia is rich in silicaceous rocks, DE samples
from this broader location have been evaluated only in one location with respect to their
insecticidal value to date. The results of the present study indicate that the DE deposits from
this region should be further evaluated as sources for commercially exploitable DEs.
However, the physical properties of these DE deposits should be further investigated with the
view to produce DE formulations that could be effective at low doses and by this means,
minimize their negative effect on grain quality.
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Acknowledgements
This study was supported by the Multilateral Research Project “Development of a non-toxic,
ecologically compatible, natural resource based insecticide from diatomaceous earth deposits
of South Eastern Europe to control stored-product insect pests” (SEE-ERA.NET; Ref. Nr 06-
1000031-9902).
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Table 1. Tested DEs and the weight (%, w/w) of 3 fractions of DE particles. Origin
(geographic location) of each DE is mentioned inside parenthesis.
DE Sample<45 microns
(%)
45-150 microns
(%)
0-150 microns
(%)
Elassona 1. (Greece) 50 50 100.0
Elassona 2 (Greece) 55 45 100.0
Kriti (Greece) 60 40 100.0
Begora (Greece) 50 50 100.0
Kolubara 516 (Serbia) 40 60 100.0
Kolubara 517 (Serbia) 60 40 100.0
Kolubara 518 (Serbia) 75 25 100.0
Vranje 3112107 (Serbia) 40 60 100.0
Vranje (Serbia) 40 60 100.0
Serbia lower (Serbia) 60 40 100.0
Kolubara middle (Serbia) 65 35 100.0
Serbia upper (Serbia) 70 30 100.0
Slovenia (Slovenia) 30 70 100.0
SilicoSec >90 <10 100.0
13
Table 2. Mean ± (SE) mortality, corrected by Abbott’s formula, of C. ferrugineus adults previously exposed for 24h and 6d to wheat treated with
different fractions of DEs at 600 ppm. Within each exposure interval, means in the same column followed by the same uppercase letter are not
significantly different at P<0.05; Within each exposure interval, means in the same row followed by the same lowercase letter are not significantly
different at P<0.05; Uppercase letters show differences between the tested DEs; Lowercase letters show differences between the different fractions of
a specific DE; For the tested DEs, df= 13,125; For the tested particle sizes df=2, 26.Exposure interval
24h 6d
DE sample 0-45 microns 45-150 microns 0-150 microns 0-45 microns 45-150 microns 0-150 microns
Begora 37.1 ± 2.1 Fa 16.9 ± 1.8 EFb 34.4 ± 0.8 EFa 91.7 ± 1.9 BCa 75.3 ± 1.6 Bb 86.2 ± 2.1 Ba
Elassona 1 98.7 ± 0.6 Aa 67.3 ± 4.1 Bb 90.7 ± 2.7 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
Elassona 2 49.1 ± 1.1 Ea 7.6 ± 0.9 FGc 38.9 ± 1.7 EFb 100.0 ± 0.0 Aa 45.6 ± 2.5 Db 100.0 ± 0.0 Aa
Kolubara 516 12.4 ± 1.5 GHa 3.3 ± 0.5 Gb 13.6 ± 1.9 HIa 70.2 ± 1.8 Da 42.2 ± 2.0 Db 63.1 ± 3.5 Da
Kolubara 517 21.3 ± 1.1 Gab 17.6 ± 1.1 EFb 22.4 ± 1.1 FGHa 100.0 ± 0.0 Aa 97.3 ±1.9 Aa 100.0 ± 0.0 Aa
Kolubara 518 74.0 ± 3.2 Ca 28.7 ± 3.1 DEb 69.1 ±3.9 BCDa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
Kolubara middle 52.7 ± 4.4 DEa 49.3 ± 5.7 Ca 47.8 ± 4.4 CDa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
Kriti 90.7 ± 1.1 ABa 28.7 ± 1.3 DEb 87.6 ± 1.9 Ba 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
Serbia lower 6.9 ± 1.1 Ha 1.1 ± 0.6 Gb 7.11 ± 0.7 Ia 86.2 ± 3.5 Ca 47.1 ± 2.6 Dc 72.9 ± 4.0 Cb
Serbia upper 61.6 ± 1.8 Da 17.1 ± 1.9 EFc 47.6 ±1.5 DEb 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
Slovenia 82.9 ± 2.2 BCa 30.0 3.9 Dc 65.1 ± 3.8 BCb 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
Vranje 16.1 ± 1.1 GHa 10.9 ± 1.8 FGa 11.8 ± 1.7 GHIa 94.2 ± 0.8 Ba 68.0 ± 2.6 Cb 74.0 ± 1.6 Cb
Vranje3112107 54.2 ± 2.5 DEa 4.4 ± 0.7 Gc 20.2 ± 2.5 FGb 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
Silicosec 100.0 ± 0.0 Aa 99.6 ± 0.3 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
14
Table 3. Mean ± (SE) mortality, corrected by Abbott’s formula, of S. oryzae adults previously exposed for 24h and 6d to wheat treated with different
fractions of DEs at 600 ppm. Within each exposure interval, means in the same column followed by the same uppercase letter are not significantly
different at P<0.05; Within each exposure interval, means in the same row followed by the same lowercase letter are not significantly different at
P<0.05; Uppercase letters show differences between the tested DEs; Lowercase letters show differences between the different fractions of a specific
DE; For the tested DEs, df= 13,125; For the tested particle sizes df=2, 26.Exposure interval
24h 6d
DE sample 0-45 microns 45-150 microns 0-150 microns 0-45 microns 45-150 microns 0-150 microns
Begora 0.0 ± 0.0 Ea 0.0 ± 0.0 Ca 0.0 ± 0.0 Ga 19.6 ± 2.1 Ha 4.6 ± 1.0 Gc 12.4 ± 1.4 Ib
Elassona 1 8.0 ± 1.1 Ba 4.7 ± 0.5 Bb 4.9 ± 0.7 Bb 89.8 ±1.9 Ba 72.0 ± 3.3 Cb 80.0 ± 2.3 CDb
Elassona 2 4.0 ± 0.6 CDa 0.0 ± 0.0 Cc 2.0 ± 0.5 DEFb 28.2 ± 2.0 Ga 18.9 ± 1.5 Fb 24.7 ± 2.2 Hab
Kolubara 516 1.8 ± 0.5 DEab 0.7 ±0.3 Cb 2.7 ± 0.3 EFGa 41.8 ± 0.7 Fa 19.1 ± 1.4 Fc 24.4 ± 1.4 GHb
Kolubara 517 5.3 ± 0.8 BCa 1.9 ± 0.7 BCb 4.2 ± 0.8 BCDab 66.1 ± 1.2 Da 45.4 ± 1.2 Ec 5.3 ± 1.8 Fb
Kolubara 518 4.2 ± 0.4 CDa 2.2 ± 1.2 BCa 4.0 ±0.3 CDEa 97.6 ± 0.4 Aa 89.6 ± 1.3 Bb 94.7 ± 1.5 ABa
Kolubara middle 2.4 ± 0.3 CDEa 0.0 ± 0.0 Cb 0.0 ± 0.0 Gb 98.2 ± 0.6 Aa 74.0 ± 1.6 Cb 93.6 ± 1.8 BCa
Kriti 5.1 ± 0.7 BCab 3.8 ± 0.2 Bb 6.9 ± 1.0 BCa 42.4 ± 0.7 Fa 22.2 ± 1.0 Fc 29.3 ± 1.9 GHb
Serbia lower 2.4 ± 0.3 CDEa 0.0 ± 0.0 Cb 1.8 ± 0.8 FGa 51.1 ±1.5 Ea 14.2 ±1.8 Fc 44.0 ±2.3 Gb
Serbia upper 4.4 ± 0.6 CDa 2.7 ± 0.8 BCa 3.6 ± 0.7 FGa 76.2 ± 2.1 Ca 56.4 ± 3.1 Db 67.6 ± 2.5 Ea
Slovenia 1.3 ± 0.5 DEb 0.0 ± 0.0 Cc 2.9 ± 0.4 FGa 86.7 ± 1.5 Ba 62.0 ± 2.3 Dc 78.4 ±2.1 DEb
Vranje 0.0 ± 0.0 Ea 0.0 ± 0.0 Ca 0.0 ± 0.0 Ga 41.6 ± 1.9 Fa 20.7 ± 1.5 Fc 30.7 ± 1.7 GHb
Vranje3112107 4.2 ± 0.4 CDa 1.8 ± 0.7 BCb 4.9 ± 0.4 CDEa 87.3 ±1.5 Ba 75.6 ±1.5 Cb 81.8 ± 1.8 CDa
Silicosec 21.8 ± 1.5 Aa 16.9 ± 1.5 Ab 22.4 ± 1.5 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
15
Table 4. Mean ± (SE) mortality, corrected by Abbott’s formula, of R. dominica adults previously exposed for 24h and 6d to wheat treated with
different fractions of DEs at 600 ppm. Within each exposure interval, means in the same column followed by the same uppercase letter are not
significantly different at P<0.05; Within each exposure interval, means in the same row followed by the same lowercase letter are not significantly
different at P<0.05; Uppercase letters show differences between the tested DEs; Lowercase letters show differences between the different fractions of
a specific DE; For the tested DEs, df= 13,125; For the tested particle sizes df=2, 26.Exposure interval
24h 6d
DE sample 0-45 microns 45-150 microns 0-150 microns 0-45 microns 45-150 microns 0-150 microns
Begora 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 30.0 ± 2.2 Ea 8.4 ± 2.1 EFGHb 11.3 ±1.1 DEb
Elassona 1 2.2 ± 0.2 BCa 0.0 ± 0.0 Bc 1.3 ± 0.3 BCb 63.8 ± 3.3 Ba 12.4 ±2.3 CDEFb 57.1 ± 3.9 Ba
Elassona 2 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 11.1 ± 2.1 GHa 0.9 ± 0.4 Hb 8.9 ± 1.4 EFa
Kolubara 516 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 16.7 ± 0.8 FGa 10.4 ± 1.9 DEFGb 14.0 ± 2.0 DEFab
Kolubara 517 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 22.0 ± 1.2 EFa 9.3 ±1.5 DEFGHb 14.2 ±1.7 DEb
Kolubara 518 2.4 ± 0.3 BCa 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 54.0 ± 2.3 CDa 31.1 ± 3.1 Bc 41.3 ± 2.5 Bb
Kolubara middle 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 60.2 ± 3.2 BCa 28.2 ± 2.5 Bb 54.2 ±1.6 Ba
Kriti 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 26.2 ± 1.5 Ea 3.3 ± 0.9 GHb 21.3 ± 2.0 DEa
Serbia lower 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 4.4 ± 1.0 Ha 0.9 ± 0.5 Hb 4.4 ± 1.6 Fa
Serbia upper 3.1 ± 0.4 Ba 0.0 ± 0.0 Bb 0.0 ± 0.0 Cb 44.9 ± 2.8 Da 16.2 ± 1.9 CDEb 23.3 ± 2.3 Cb
Slovenia 3.1 ± 0.5 Ba 0.0 ±0.0 Bb 2.7 ±0.3 Ba 47.1 ± 0.5 Da 17.3 ± 2.3 CDc 26.4 ± 2.1 Cb
Vranje 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 14.0 ± 1.2 FGa 6.0 ± 0.8 FGHc 9.6 ± 0.8 EFb
Vranje3112107 0.0 ± 0.0 Ca 0.0 ± 0.0 Ba 0.0 ± 0.0 Ca 28.9 ± 1.9 Ea 19.3 ± 1.3 Cb 22.2 ± 3.0 CDab
Silicosec 18.1 ± 2.1 Aa 11.1 ± 1.3 Ab 16.0 ± 0.9 Aab 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa 100.0 ± 0.0 Aa
16
Fig.1. Correlation (Pearson’s coefficient) between the efficacy of the base-DE samples and the
proportion in small particles (<45 microns) of the base-DE samples, against adults of C.
ferrugineus, S. oryzae and R. dominica at two exposure intervals.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
24h 6d
C. ferrugineus
S. oryzae
R. dominica
Exposure interval
Pear
son'
s coe
ffic
ient
17